SUMMARY: The emergence of the SARS-CoV-2 coronavirus (CoV) highlighted our lack of preparedness, and has emphasized the importance of rapidly building capacity in the development of reagents, tools, diagnostics and therapeutics for SARS-CoV-2 and related CoVs with pandemic potential. An immediate goal is to produce a vaccine which can elicit rapid, high-level protective immunity against SARS-CoV-2, ideally following a single- shot. Several candidate vaccines have now advanced into clinical trials. However, a longer term goal is to investigate the possibility for a ?universal? CoV vaccine, a vaccine or prime:boost vaccination regimen which provides durable and broadly cross-reactive immunity against CoVs with high potential for spillover from bats. The CoV surface spike (S) glycoprotein is a major target for neutralizing antibodies (NAbs) and T cells, and is an attractive target for vaccine design. NAbs which target the receptor binding domain (RBD) confer protection, but are usually strain-specific and lack breadth. The existence of broadly reactive, protective epitopes outside of the RBD are not well-characterized. Therefore, vaccines which compare full length or truncated, stabilized variants of the S immunogen, could shed some light into potential correlates of protection. Another important concern is disease enhancement, which has been observed for related CoV vaccines using selected vaccine delivery platforms such as the whole-inactivated virus (WIV) vaccine. This has been associated with a Th2- biased immune response and to overcome this issue, CoV vaccines should elicit a largely Th1 biased response. Therefore, studies which aim to compare the phenotype of immunity elicited by different vaccine platforms, and to different variants of the S immunogen, could help to better understand which components of the immune response are optimal in mediating protection, without the risk of immunopathology upon infection. We will develop a potently immunogenic, optimized vaccine platform for SARS-CoV-2 using three approaches. (1) Firstly, we will engineer SARS-CoV-2 S in several different forms, a full-length immunogen, a secreted stabilized pre-fusion form or the RBD domain alone. (2) Secondly, we will augment or broaden immune recognition of pre-fusion S by targeting it to host-derived extracellular vesicles (EVs) including exosomes in vivo, by generating fusion-Ag constructs which tether Ag to a protein domain highly enriched in exosomes. Exosomes are nano-sized EVs shown to play important roles in cell:cell communication and in the regulation of immune responses, due to their ability to present Ag to T- and B-cells. (3) Finally, we will develop non-replicating, rare species adenoviral (Ad) vectored vaccines which have established protocols for rapid clinical manufacturing and regulatory approval, can be thermostabilized with minimal losses to immunogenicity and have demonstrated safety in human clinical trials. This study will comprehensively evaluate and phenotype the magnitude and profile of SARS-CoV-2 vaccines in single-shot regimens. These data will provide valuable information for the design of subsequent prime:boost regimens and for challenge experiments in the future.